1. Papillomavirus infection and viral gene expression Human papillomavirus type 16 (HPV16) or 18 (HPV18) infection, acquired primarily via sexual transmission, is widely recognized as a leading cause of cervical and anal cancer and thus, are classified as being oncogenic. Infection with oncogenic HPV in other tissues could also lead to development of cancer. For example, we recently demonstrated that colorectal HPV infection, particularly with oncogenic HPVs, is common in patients (51%) with colorectal cancer. High prevalence and incidence of cervical HPV infection has been observed among HIV-positive and immunodeficient women. Oncogenic HPV16 DNA was also found in the peripheral blood mononuclear cells of transfusion-acquired pediatric HIV patients, but also from some healthy blood donors. Cervical cancer has been the most common malignancy among women with AIDS in both Europe and the United States. Two viral oncoproteins in particular, E6 and E7, of HPV16 and HPV18 are involved in cervical carcinogenesis and are known to inactivate cellular tumor suppressor proteins p53 and pRb, respectively. HPV16 and HPV18 E6 and E7 are transcribed as a single bicistronic RNA bearing 3 exons and 2 introns, with the intron 1 in the E6 coding region. Various studies, including those from our laboratory, have demonstrated that splicing of intron 1 in the HPV16 E6E7 pre-mRNA is highly efficient with the majority of the transcripts in cancer tissues and cervical cancer cell lines encoding E6*I, a spliced product without intron 1. It is our hypothesis that the E6 is expressed from a small portion of the bicistronic RNAs, without splicing of the intron 1, which has raised several important questions: 1. Why is efficient splicing of intron 1 in HPV16 or HPV18 E6E7 pre-mRNA needed for viral gene expression since splicing harms E6 expression? 2. How does a small portion of the total pool of E6E7 pre-mRNAs escape splicing of intron 1 and what regulates this escape? 3. How could an RNA molecule containing an intron be exported to the cytoplasm to translate E6 protein? The answers to these important questions will come from continuing our studies aimed at describing the mechanisms involved in this RNA splicing regulation. We have successfully demonstrated that intron 1 splicing subjects the RNA molecule to exon definition (size) by a cap structure on the RNA 5' end. Strong evidence, from multiple approaches in transient transfection and in cervical cancer-derived HPV16+ and HPV18+ cell lines, indicate that the ability of the RNA molecule to escape splicing of intron 1 results in production of unspliced E6 mRNA to subsequently encode viral oncoprotein E6, whereas splicing of the intron promotes E7 oncoprotein expression. We also identified several E6 and E7-specific siRNAs for selective post-transcriptional silencing of the two viral oncogenes. We further demonstrated that, although those siRNAs have therapeutic potentials for acute treatment, long-term delivery of the E6 or E7-specific siRNAs might induce cellular resistance to siRNA function. Recently, we have also demonstrated that some high-risk HPV proteins, such as HPV16 E2 and E6, are RNA binding proteins in vitro and that they interact with the cellular splicing factors responsible for regulating the splicing of HPV16 E6E7 pre-mRNAs. 2. KSHV Gene expression and post-transcriptional regulation KSHV is a lymphotropic DNA tumor virus that induces Kaposis sarcoma (KS), body cavity-based B-cell lymphoma, and multicentric Castlemans disease. Among those malignancies, KS occurs frequently in patients infected with HIV. Latent KSHV infection in KS lesions and PEL-derived B cells features the highly restricted expression of only five viral genes. The lytic KSHV infection can be induced by chemicals in PEL-derived B cells with latent KSHV infection. In this lytic switch, a KSHV transactivator, ORF50, is absolutely required. ORF50 is an immediate-early gene transcribed as a polycistronic RNA with 5 exons and 4 introns. ORF50 is positioned in the virus genome with both K8 (an early gene encoding a K-bZIP protein) and K8.1 (a late gene encoding a viral envelope glycoprotein) and shares a single polyadenylation site downstream of K8.1 coding region. Accordingly, the transcripts of the three genes overlap each other and undergo extensive RNA splicing. However, there are two major isoforms of spliced RNA products, &#945; (exclusion of K8 intron 2 or ORF50 intron 3) and &#946; (inclusion of K8 intron 2 or ORF50 intron 3) in KSHV lytic infection. Based on our complete profiling of the transcription and splicing of ORF50, K8 and K8.1, we demonstrated that KSHV K8&#946; is derived from a splicing intermediate and antagonizes K8&#945;-mediated induction of p21 and p53 and blocks K8&#945;-CDK2 interaction. We have also extended our investigation to KSHV ORF57, a ICP27 homolog of herpes simplex viruses involved in regulation of virus replication and also mediates viral RNA export. We demonstrated that the ORF57 is a phosphorylated nuclear protein bearing three nuclear localization signals in its N-terminus. We have also analyzed the gene structure and expression of KSHV ORF56 (viral primase), ORF57 (mRNA transcript accumulator, MTA), ORF58 (EB virus BMRF2 homology) and ORF59 (viral DNA polymerase processing factor) and demonstrated that both ORF56 and ORF59 are expressed as bicistronic RNAs that subject to ORF57 up-regulation. Recently, we demonstrated KSHV ORF57 as a viral splicing factor in promoting splicing of intron-containing viral RNAs. Works are in progress to understand the mechanisms on how KSHV ORF57 might regulate viral gene expression post-transcriptionally